Central nervous system (CNS) inflammation is a hallmark of HIV neuropathogenesis in untreated advanced AIDS. During systemically suppressive combination antiretroviral therapy (cART), abnormal inflammation persists, and associates with mild neurological impairment and, in dramatic form, with progressive neurologic disease in symptomatic cerebrospinal fluid (CSF) HIV escape and CD8+ T cell encephalitis. Defining neuroinflammation in exquisite detail, including rare and/or novel populations that distinguish HIV infection during cART, has the potential to provide critical targets for therapeutic intervention for residual neurologic impairment during HIV treatment. The proposed studies capitalize on recent dramatic expansion of technologies and understanding of microfluidics, molecular barcoding, and sequencing to facilitate the precise, unbiased, and high throughput sequencing of RNA expressed in single cells. Thousands of individuals cells in a given tissue type can now be profiled in detail to understand the cellular composition of healthy tissues, and to begin to unravel the cellular disruptions present in disease. We have recently successfully applied massively-parallel single-cell RNA sequencing to transcriptionally profile thousands of cells derived from the CSF at the single cell level. Our preliminary studies demonstrate application of Seq-Well, a portable, low-cost platform for single cell RNA- sequencing designed to be compatible with low-input clinical samples, to analysis of CSF and blood from individuals with well-treated HIV and HIV-uninfected controls. In the current R21 proposal, we seek to apply this breakthrough methodology followed by advanced bioinformatics analysis to demonstrate the utility of single cell RNA sequencing of CSF to study pathogenesis and mechanisms of HIV related injury in the CNS in individuals with HIV on CART. Large datasets generated through high throughput sequencing require meticulous and expert analyses. Our planned thorough bioinformatics approach will provide unprecedented depth of insight into complex processes, and will demonstrate the potential of single cell RNA sequencing of CSF to help to unravel the complexities of neuroinflammation relevant to CNS HIV and numerous other neurological disorders.